EXTH-53. IMPACT OF EGFRvIII AND PTEN DELETION MUTATIONS ON RESPONSE OF Ink4a/Arf-NULL MURINE ASTROCYTES TO EGFR TYROSINE KINASE INHIBITORS

摘要

Epidermal growth factor receptor (EGFR) represents the most attractive target for personalized medicine of glioblastoma (GBM) due to its frequency in and specificity for the disease, and the availability of multiple inhibitors that target its tyrosine kinase domain. Despite its therapeutic attractiveness, several EGFR-targeted tyrosine kinase inhibitors (TKI) have failed clinically, in part due to multiple molecular mechanisms of resistance. To dissect the genetic contributions to EGFR TKI resistance, we examined the sensitivity of Ink4a/Arf (CDKN2A)-null murine astrocytes engineered to harbor EGFRvIII, the most common activating mutation in GBM where exons 2–7 of the extracellular domain are deleted, to a panel of first (gefitinib, erlotinib), second (lapatinib, afatinib), and third (AZD3759) generation EGFR TKI in vitro. Moreover, we examined the role of PTEN deletion mutations on drug sensitivity. We found that EGFRvIII conferred sensitivity to 4/5 drugs (ΔIC50 1.5-6.5-fold) in the presence of wild-type Pten and 5/5 (ΔIC50 1.5-78.0-fold) when Pten was deleted. In contrast, Pten deletion conferred resistance to 4/5 drugs (ΔIC50 5.6-17.7-fold) in Ink4a/Arf-null astrocytes with wild-type Egfr and 2/5 (ΔIC50 1.4-5.4-fold) when these cells harbored EGFRvIII. Moreover, Ink4a/Arf-null, EGFRvIII-mutated astrocytes developed cross resistance to the other 4 TKI (ΔIC50 2.6-11.0-fold) when grown continuously in the presence of increasing gefitinib (≤ 2 µM). We have previously shown that dynamic kinome responses may be responsible for TKI resistance. Therefore, we are currently using a chemical proteomics method consisting of multiplex inhibitor beads and mass spectrometry (MIB-MS) to assess the functional state of the kinomes of these non-germline genetically engineered mouse (nGEM) models en masse, both in the presence (dynamic profiling) and absence (baseline profiling) of EGFR TKI. Thus, functional kinome analysis using targeted EGFR TKI and MIB-MS will help define the kinase networks required for EGFRvIII-driven GBM pathogenesis and may aid in the identification of novel treatment combinations.